Nevertheless, there is no data about the role of mosGCTL-1 as a transmission-blocking vaccine candidate in Culex pipiens, the most abundant Culicinae mosquito in temperate regions. Methods: Adult female Cx. pipiens mosquitoes were experimentally infected with a WNV infectious blood meal, and the effect of rabbit anti-rmosGCTL-1 antibodies on virus replication was evaluated. Additionally, in silico studies such as the prediction of protein structure, homology modeling, and molecular interactions were carried out. Results: We showed a 30% blocking activity of Cx. pipiens mosGCTL-1 polyclonal antibodies (compared to the 10% in the control group) with a decrease in infection rates in mosquitoes at day 5 post-infection, suggesting that there may be other proteins in the midgut of Cx. pipiens that could act as cooperative-receptors for WNV. In addition, docking results revealed that WNV binds with high affinity, to the Culex mosquito lectin receptors. Conclusions: Our results do not support the idea that mosGCTL-1 of Cx. pipiens primarily interacts with WNV to promote viral infection, suggesting that other mosGCTLs may act as primary infection factors in Cx. pipiens.Background: Mosquito galactose-specific C-type lectins (mosGCTLs), such as mosGCTL-1, act as ligands to facilitate the invasion of flaviviruses like West Nile virus (WNV). WNV interacts with the  Arthropod-borne diseases contribute to the epidemics that disrupt health security , represeOnly a few vaccines related to mosquito-borne diseases such as Dengvaxia against DENV and the 17D against yellow fever virus (YFV) are available ,6. ContrmosGCTL-1), and interactions between WNV and mosGCTL-1 are critical for infection of Aedes aegypti and Culex quinquefasciatus. In that study, generated antibodies against mosGCTL-1 of Ae. aegypti in rabbits inhibited WNV infection. Further, infection of Cx. quinquefasciatus (CPIJ010995) to WNV, resulted in up-regulation of mosGCTL-1 homolog in this mosquito [In 2010, Cheng et al., reported that WNV induces the expression of mosquito galactose-specific C-type lectin-1  gene of this strain was submitted to the GenBank under the KY646203 accession number). Nevertheless, Cx. quinquefasciatus was not identified in all the collected and examined Culex mosquito specimens.Morphological identification and molecular analysis revealed that the collected mosGCTL-1 sequences of Culex species, a set of primers and an optimized amplification program were considered as described. The mosGCTL-1 gene of Cx. pipiens encodes 159 amino acid residues; the length of intron for this gene in Cx. pipiens was 61 bp (MF361860 and MF361861 accession numbers), which sharing 93% of identity with the mosGCTL-1 of Cx. quinquefasciatus (WGS: AAWU01017778) mosGCTL-1 , (15/20) in comparison to the control group (18/20), (16/20) respectively .Cx. pipiens and other species indicated that this protein has very conserved residues, and it has predicted with more than 85% accuracy that Culex species have close root  epitopes with MHC-II using the NetMHCII server showed that the highest affinities among the evaluated CTLM peptides belonged to the HLA-DQA10102-DQB10602. Presentation capability by the common MHC-II alleles, which is determined by the affinity of the target peptides, is presented in 2+ has special importance in the structure of MBPs and some of them have two calcium ions in their structure. One of them, which is deployed near the loop and named Ca2+ site 1, is auxiliary and induces the compacted loop structure [2+ site 2, which is called principal Ca2+, is very conserved among the all C-type lectin-like domains and plays an important role in binding to the sugar ligands. This calcium interacts directly with the β4 strand, the extended loop, and sugar ligands. Interestingly, the upper part of the loop of our target protein, which contains the residues from PHE 91 to ASP112, is involved in this interaction. The extended part of the loop which contains residues from ASP84 to THR 90 and GLU113 to ASP118 are named regions I and II, respectively. This section contains a set of aromatic and hydrophobic amino acids such as PHE 91, TRP 93, TRP 105, PRO110 as well as TRP134 of the β4 strand which participate in the ligand interactions [2+ such as GLU 109, ASN 111, and ASP 112, participate in interactions with ligand. Typically, calcium has eight coordination: five coordination with amino acids, and one bond with the water molecule. The presence of Ca2+ and the formation of suitable hydrogen bonds with amino acids provide the base for the binding of sugar molecules to this site. In the process of hydrogen bond formation, electron exchange takes place from an electron donor, which could be one sugar OH group, to the NH2 group of asparagine. Therefore, the presence of suitable amino acids as an electron acceptor is critical for hydrogen bond formation and binding of sugar ligands [Based on the results of the SWISS-MODEL server, which was confirmed using the Phyre 2, C-type carbohydrate-recognition domain (CRD-4) from the macrophage mannose receptor; chain B (PDB accession No: 1EGI.B) had the highest structural identity 25.2%) to the target protein with a 97.5% confidence. Therefore this molecule was used as a reference molecule for beyond analysis. Superimposition of the reference molecule (1EGI) and CTLM showed that these two molecules were structurally 23.70% identical to each other, overall root-mean-square deviation (RMSD) was 0.567, and Q-score was 0.781. The primary structure of the model belonged to a family of calcium-dependent carbohydrate-binding proteins. The structure of this molecule consists of two α-helixes, five antiparallel ß-strands, and a wide flexible loop. Disulfide bonds due to the two cysteine residues stabilize the three-stranded antiparallel sheets which are conserved in the other C-type lectins such as lithostathine , tetrane.2% to thtructure . Howeverractions . Moreove ligands . On the  ligands   between the receptor and the ligand was compared. Interestingly, this evaluation confirmed the previous docking results as well .Culex pipiens is a primary vector of WNV, maintaining local enzootic transmission among avian species [pipiens and molestus [Cx. pipiens biologic form are capable of laying eggs without a blood meal in their first gonotrophic cycle, but those of the Cx. pipiens biologic form pipiens require a blood meal for reproduction [Cx. pipiens belonged to pipiens form. We also could not find any Cx. quinquefasciatus species in the collected samples. This finding is accordant with the former investigations, which had reported the presence of Cx. quinquefasciatus in southern to central areas of the country [ species ,31. Thismolestus , and is molestus . The femoduction . In our  country .Ae. aegypti [mosGCTL-1 and a protein tyrosine phosphatase (PTP) of Ae. aegypti), named mosPTP-1 , act as a part of the same pathway which is important for infection of mosquitoes to the WNV. The mosPTP-1 participates in virus endocytosis, but its role as an attachment factor has remained unknown yet [Ae. aegypti and reduced its vector competence. It has been revealed that silencing of the mosGCTL-1 does not influence DENV infection in Ae. aegypti which suggests that the product of this gene only facilitates WNV invasion [It has been shown that many midgut proteins in mosquitoes are involved in interacting with arboviruses . For exa aegypti , suggest aegypti . Cheng enown yet . It has invasion .Cx. pipiens mosGCTL-1 was characterized using the primers which were designed with bioinformatics tools. The mosGCTL-1 sequences were differed by 93% between the Cx. pipiens and Cx. quinquefasciatus (WGS: AAWU01017778). The intron length of the mosGCTL-1 was varied and depends on the mosquito species: 61 bp in our study in comparison to 41 bp in the Cx. quinquefasciatus [Cx. pipiens mosGCTL-1 encodes 159 amino acid residues compared to the 165 in Cx. quinquefasciatus [mosGCTL-1 interacts with arboviruses, differences in the amino acid residues in different mosquitoes may affect the interactions of flaviviruses with this protein and consequently, these differences in the sequence may result in a structural difference and affect the vector competence of these species.In our study, asciatus . Furtherasciatus . RegardiAe. aegypti that resulted in WNV infection blockage with the rmosGCTL-1 antibodies as well as the up-regulation of Cx. quinquefasciatus mosGCTL-1 by WNV infection [Our findings suggest that our produced polyclonal antibodies against mosGCTL-1 cannot efficiently block WNV compared to the previously performed study, which was carried out on nfection .Culex pipiens C-type lectins revealed that this protein has conserved residues that represent its key role in different species. The juxtaposition of different species of Culex and even the closeness of Aedes and Drosophila in the phylogenic tree confirms this idea  and the WNV was significantly lower than the reference lectin (1EGI). In other words, the binding of Cx. pipiens lectin is much more effective to the virus  located in Golestan province, northeast of Iran. Mosquitoes were reared in the national insectary of Iran, malaria and vector research group (MVRG), and Pasteur Institute of Iran . Larvae were identified using the key of Azari-Hamidian and Harbach [Cx. pipiens and Cx. quinquefasciatus species [Cx pipiens species were reared under standardized conditions of the insectary . Larvae were placed in dechlorinated water supplemented with fish powdered food. Emerging adults were daily collected and transferred into cages and they were fed with a 10% glucose solution.According to the standard dipping technique (350 mL dipper),  Harbach . MorpholCx. quinquefasciatus and Ae. aegypti mosGCTL-1  [Cx. pipiens mosGCTL-1. As no match was found in BLAST Nucleotide collection (nr/nt) database (http://blast.ncbi.nlm.nih.gov/Blast.cgi), other databases of GenBank including Transcriptome Shotgun Assembly (TSA), Sequence Read Archive (SRA) and Whole-Genome Shotgun Contigs (WGS) databases were also used . Primers were designed using the Gene Runner software .The sequences of ctively) ) were usCx. pipiens were extracted from the midgut and salivary glands of female mosquitoes. Then, RT-PCR was carried out using the QIAGEN® kits . PCR reaction mixture contained 400 nM (each) primers, 1 unit of Taq DNA polymerase, 0.2 mM (each) dNTPs, 0.001% gelatin, 2.5 µL of reaction buffer, 1.5 mM MgCl2, and 1 µL of RT reaction product (for RT-PCR) in a total volume of 25 µL. Additionally, 100 ng of extracted DNA was used as the template to amplify the nucleotides containing the intron. A set of primers for mosGCTL-1 amplification were designed: mos-F: 5′-CGACTTCGACTCAACATAAGCAG-3′ and mos-R: 5′-CTAAAACGGCGCCACACAATCG-3′. The optimized program was set as initial denaturation at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 35 s, annealing at 58 °C for 35 s, and extension at 72 °C for 40 s, and an additional final extension at 72 °C for 10 min.In the current investigation, total RNA and DNA of the reared ®, Seoul, South Korea) and sequenced with an ABI 310 DNA sequencer machine . Molecular and phylogenetic designing and analysis were carried out based on the bioinformatics tools such as Gene Runner (version 5.1.01d Beta), Chromas (version 2.6), and Mega7 (version 7.0) [The PCR products were visualized on the agarose gel, and expected bands were recovered with a gel extraction kit (GeneAllion 7.0) .Cx. pipiens mosGCTL-1 gene was cloned into the pET23a-plasmid using the BamHI and HindIII restriction sites and expressed in Escherichia coli BL21 (DE3). The E. coli BL21-pET23a-Cx. pipiens mosGCTL-1-clone was grown in Terrific Broth (TB). When optical density (OD) at 600 nm was equal to 0.6–0.8, the expression was induced by adding the isopropyl-beta-d-1-thiogalactopyranoside , and incubated for overnight.Having the sequencing results, the following primers: mosmrnaF: 5′-GGATCCATATGTTGTCGAAAAGAAGTCTAGGAG-3′ and mosmrnaR: 5′-AAGCTTAAACGGCGCCACACAATC-3′ were designed for cloning process under the optimized amplification program: initial denaturation at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 35 s, and an additional final extension at 72 °C for 10 min. The PCR product of 595nm. The produced protein was analyzed after electrophoresis in 15% SDS–polyacrylamide gel (SDS-PAGE). Western blot assay was performed using the mouse anti-His antibody  as the primary antibody, and Peroxidase conjugated anti-mouse IgG antibody  as the secondary antibody to confirm the purity of the rmosGCTL-1 , and the eluates containing rmosGCTL-1 were desalted using Econo-Pac 10 DG columns . Bradford protein assay was used to measure the concentration of rmosGCTL-1 protein using a spectrophotometer  at ODosGCTL-1 . To prodCx. pipiens were shipped under the optimal condition to the insectary of arboviruses and insect vectors unit, Institute Pasteur of Paris for WNV experimental infections as described before [Cx. pipiens were placed into the boxes, starved for 48 h in biosafety level 3 insectary  and then were allowed to feed for 20 min through a pig intestine membrane covering the base of a capsule of the feeding system  containing the blood–virus mixture which was maintained at 37 °C [−3 M. Virus titer in the blood-meal was at 107.3 pfu/mL. After feeding, the fully engorged females were transferred into the cardboard containers and maintained with 10% sucrose at 28 ± 1 °C for 7 days. To evaluate the blocking efficiency of the anti-rmosGCTL-1 antibodies, the infection rate and Log RNA copies/body were assessed in the control and test groups at 0 (3 samples), 2 (20 samples), 5 (20 samples), and 7 (20 samples) days post-infection (dpi) by RT-qPCR method. Total RNA of the 126 infected mosquito whole body  were extracted using the Macherey-Nagel NucleoSpin® RNA extraction kit  to estimate the viral infection rate. Total WNV RNA was quantified by RT-qPCR using the Bio-Rad CFX96™ Real-Time PCR Detection System and Power SYBR® Green RNA-to-CT 1-Step kit. For each reaction, 10.55 µL of distilled water, 12.5 µL of buffer 2X, 0.375 µL (10mM) of each designed forward and reverse primers (WN175up: 5′-GTGTTGGCTCTCTTGGCGTT-3′ and WN259low: 5′-AGGTGTTTCATCGCTGTTTG-3′), 0.2 µL of mix-enzyme 125X, and 1 µL of RNA were used. The reverse transcription reaction was performed at 48 °C for 30 min. The qPCR conditions were 95 °C for 10 min, followed by 40 amplification cycles of 95 °C for 15 s and 60 °C for 1 min. Additionally, melt curve assay (65.0 °C to 92.0 °C: Increment of 0.5 °C 0:05) was carried out. For each run, the number of WNV RNA copies was calculated by absolute quantitation using a standard curve.Due to the unavailability of the BSL3 facility in Pasteur Institute of Iran and in collaboration with Institut Pasteur in Paris, after signing a biological material agreement; the egg rafts of d before . Seven-tat 37 °C . The infat 37 °C  and an eFirst, by referring to the NCBI, the protein BLAST tool was used to search similar proteins to our query. Some subjects with higher similarity and scores, which were from different species, were selected to perform multiple alignments using the Clustal Omega  based onProper presentation and effective stimulation of the immune system are the most important factors which should be considered for selecting and evaluating a vaccine candidate molecule. For this purpose, the antigen-presenting cells break down the antigenic epitopes into suitable fragments. Depend on the features of an antigen and presenting cells, antigenic peptides are broken into different pieces. These fragments are delivered to the effector immune cells by the major histocompatibility complex (MHC) system. Therefore, the NetMHCII 2.2 server was used to evaluate the potential properties of presenting the recombinant C-type lectin with the common MHC-II alleles in Iran. Iranian’s common MHC-II alleles were determined according to the previously performed studies ,50. The Prediction of the 3D structure of mosGCTL-1 was performed using the SWISS-MODEL. The model design was accomplished based on the submitted primary amino acid sequence (GenBank: AUD11983.1). BLAST and HHBlits were used to find the most suitable patterns in the template library (SMTL version 2020-12-16) and maximum structure compatibility, respectively. Model construction was carried out using the ProMod3. Simulation of CTLM was done using the conserved residues and coordinating between the target sequence and the template. In the case of failed simulation by ProMod3, an alternative method, PROMOD-II, was used. Quality of estimated model and the final accuracy was evaluated using the QMEAN and GMQE, respectively ,54,55,56Cx. pipiens, one of the most appropriate and common technics is matching the target molecule with structurally characterized molecules. To achieve this goal, the C-type carbohydrate-recognition domain from the macrophage mannose receptor (PDB accession No. 1EGI) was used as the reference molecule. Predicted structure validation, superimposition, and molecular docking could be used for confirming the predicted structure. In molecular docking, target and reference molecules interact with a known molecule as ligand and the strength of the interactions are compared between the two molecules. In this study, to verify the predicted structure for the target molecule, protein–protein docking was used for the discovery of the receptor-ligand kinetics and responsible residues. According to the previously performed studies, l-fructose and d-mannose act as high-affinity ligands for the C-type lectin receptors [l- fructose molecule (PDB accession No. 1K12) was used as a high affinity ligand. Protein-ligand docking was done using the Rosetta online server which is based on the scoring functions and search methodologies driven from the statistical analysis of the PDB. In this method, Metropolis Monte Carlo algorithm for identifying the binding sites and some other search strategies such as geometric hashing (FlexX), genetic algorithms (GOLD), systematic sampling (Glide), and physics-based force fields (Dock) are used for calculation and analysis [To evaluate an unknown molecule and its related interactions such as mosGCTL-1 of eceptors . Therefoanalysis ,59.Next, the probable interactions between the CTLM as a receptor and WNV envelope (PDB accession N. 2I69) as ligand were evaluated and simulated to find the most appropriate binding patterns with the lowest free energy. This analysis was accomplished using the pyDockWEB server that is the rigid-body docking software. In this method, free energy calculation is based on the Coulombic electrostatics with distance-dependent dielectric constant .Culex mosquito lectin receptor. The details of these interactions such as involved residues are given in Cx. pipiens mosGCTLs presumably serve as receptors for the WNV and may act differently from its homolog in Ae. aegypti. Furthermore, additional investigations on the up-regulation of the Cx. pipiens mosGCTLs by WNV infection to develop an efficient TBV candidate are suggested. In this investigation, the rabbits were immunized with rmosGCTL-1, formulated in Freund’s adjuvant for the production of specific IgG. To improve the immunogenicity of subunit vaccines, using proper and potent adjuvant(s) could be a promising strategy to improve the pathogen-specific humoral, cellular, and functional immune responses [It could be concluded that our in silico investigation revealed that the WNV binds with high affinity to the esponses .


Saccharomyces cerevisiae cells, affected by MW-CNTs, by cell count, culture optical density and atomic force microscopy. The yeast cells were exposed towards MW-CNTs  for 24 h. A mathematical model was applied for the evaluation of relative growth and relative death rates of yeast cells. We calculated that both of the rates are two times higher in the case if yeasts were treated by 50, 100 μg/mL of MW-CNTs containing solution, comparing to that treated by 0 and 2 μg/mL c of MW-CNTs containing solution. It was determined that the MW-CNTs have some observable effect upon the incubation of the yeast cells. The viability of yeast has decreased together with MW-CNTs concentration only after 5 h of the treatment. Therefore, we predict that the MW-CNTs can be applied for the modification of yeast cells in order to improve electrical charge transfer through the yeast cell membrane and/or the cell wall.This research aimed to evaluate the toxic effect of multi-walled carbon nanotubes (MW-CNTs) on yeast cells in order to apply MW-CNTs for possible improvement of the efficiency of microbial biofuel cells. The SEM and XRD analysis suggested that here used MW-CNTs are in the range of 10–25 nm in diameter and their structure was confirmed by Raman spectroscopy. In this study, we evaluated the viability of the yeast  Aspergillus niger , and 0.3 mg/mL yeast. During voltage measurements, the external resistances were plugged in parallel to galvanostat for the assessment of power density of the complete biofuel cell.Two graphite electrodes with different materials immobilized on the electrode surface were used in biofuel cell: one only with 9,10-phenanthrenequinone (PQ) and second with PQ and MW-CNTs. The biofuel cell was made from two-electrodes: one differently modified graphite, and another—platinum electrode. They were placed in a phosphate buffer solution with 70 mM of glucose, 10 mM KTo determine the shape and size of the MW-CNTs used in the present work, the samples were scanned using the scanning electron microscope. The shape of the carbon MW-CNTs was determined by means of the data analysis of SEM measurements. The diameter of MW-CNTs was 10–25 nm, and the length was of several micrometers .θ = 25.4°; 43.1°; 54°) are present in XRD registered patterns  of 43.1°and 54° were associated with the (100), (004) diffractions of the hexagonal graphite structure —was added in the solution. PQ has the ability to cross the cell’s membrane and achieve redox centres of enzymes located in the cytoplasm of cells. K3[Fe(CN)6] takes electrons from PQ and passes them to the electrode. We registered cyclic voltammograms using PQ, and PQ/MW-CNT modified graphite electrode 6]3–/4– oxidation/reduction processes.In this research, we used the two-redox mediators-based system, which has been described in previous our research . One redlectrode . Four peElectrochemical impedance spectroscopy based measurements represent the resistivity of the layers, formed at anode surface . In case2, while for PQ-electrode maximal power was just 1.63 nW/cm2, i.e., for PQ/MW-CNT power was 69 times higher  of MW-CNTs and at rather short-lasting exposure of MW-CNTs do not significantly affect the viability and other properties of yeast cells. At higher concentrations and after longer exposure, the increase of optical density vs. MW-CNTs concentration is observed. A plot of optical density vs. incubation time dependencies, show some differences between low (0 and 2 µg/mL) and high (50 and 100 µg/mL) MW-CNTs concentrations. The steady-state conditions were observed faster for high MW-CNTs concentrations. Also, calculated relative growth and relative death rates show the difference between results registered after incubation in low and high MW-CNTs concentrations containing solutions: both rates were two-times higher for higher concentrations of MW-CNTs. The results, obtained from electrochemical measurements, enables to state that MW-CNTs are a very good candidates for the development of microbial fuel cells, because the application of MW-CNTs in anode of biofuel cell increased generated power by 69 times and generated voltage by 8 times. Therefore, we predict that MW-CNTs can be applied for the modification of the yeast cells in order to improve electrical charge transfer through the yeast cell membrane and/or cell wall. Further, our investigations in this research area will be related to the development of a strategy of how MW-CNTs can be inserted within the yeast cell wall and membrane in order to apply such modified cells in the design of microbial fuel cell.


Stigma can be defined as a negative perception of chronically ill patients by their relatives or by society, or a similar self-perception by the patients themselves. We aimed to validate the Turkish version of the Neuroquality of Life (Neuro-QoL)-Stigma Scale for neurologic diseases. -BREF quality of life scale, the Multidimensional Scale of Perceived Social Support (MSPSS), the General Self-Efficacy (GSE) scale, and the Neuro-QoL-Stigma scale.  Forms were filled out by a total of 152 randomized patients under regular follow-up in the outpatient clinic . The forms consisted of the Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), WHOQOL The internal consistency of the Neuro-QoL-Stigma scale showed Cronbach’s α coefficients of 0.95 for all groups. The mean scores of the stigma scales were 33.42 ± 13.91 (min–max: 24–87). There were strong negative correlations between high stigma scores and GSE-T, MSPSS-T, and WHOQOL-BREF, and a positive correlation with the BDI and BAI. The Turkish version of Neuro-QoL-Stigma has satisfactory content validity and high internal consistency. Neuro-QoL-Stigma is suitable for understanding stigmatization in different neurological disorders in the Turkish population. The scale is available for use at http://www.healthmeasures.net/explore-measurement-systems/neuro-qol. Stigma is a term used frequently in relation to mental disorders, but it is still uncommon to evaluate stigmatization in the context of neurological diseases (5). However, the local burden and cultural particulars of stigmatization should be further evaluated using cross-cultural comparisons.Stigmatization is the negative labeling of an individual or specific situation and this labeling results in the exclusion of the individual from society (1). Stigmatization worsens the quality of life (QoL), increases public prejudice, causes a reduction in the perception of self-worth, has negative physical and psychological consequences, and disrupts work and family life (2–4).The various forms of stigma manifest themselves as public stigma, self-stigma , and label avoidance in the DSM-V. Stigma scales target these three areas (6). Assessment of stigmatization is very important because stigmatization itself may result in delay in treatment choice and decrease in treatment quality. Stigmatization in neurological disorders has a severe effect on the patient’s family and social relationships .In Turkey, there is a limited number of studies that evaluate stigmatization in neurological disorders such as epilepsy, tension-type headache, and Parkinson disease, and there is no scale used specifically for targeting neurological disorders (9–12). Neuro-QoL is a scale developed by the National Institute of Neurological Disorders and Stroke. Neuro-QoL consists of 13 adult and 10 pediatric subgroup tests including physical, mental, and social health (13). It is a health assessment tool that is clinically and psychometrically appropriate for major neurological disorders  . The stigma scale is one of the subscales of Neuro-QoL for pediatrics and adults in both the hard copy and computerized version. The adult long version consists of 24 items and the short version consists of the first 8 items of the long version (13). Our aim was to evaluate the reliability and validity of the adult long version of the Neuro-QoL-Stigma scale in order to use it in clinical practice and research among Turkish patients. The present study was established in two stages. The first stage was the cross-cultural adaptation. A sequential approach was followed in order to obtain linguistically equivalent versions of the adult long version of the Neuro-QoL stigma scale consisting of 24 items. Semantic equivalence was achieved with two independent translations into Turkish performed by two bilingual Turkish experts and a consensus version, followed by a final back-translation performed by another translator whose native language was English and who was also fluent in Turkish.The quality analysis of the translations and the first content validity were checked by a clinician’s review and by a cognitive debriefing panel with 10 healthy literate people. After completing the questionnaires, we asked them to explain the complicated and difficult issues to the participants. Corrections were made according to suggestions deemed suitable.In the second phase, the reliability and validity tests of the Turkish version were examined. We evaluated the internal consistency reliability and construct validity of the stigma subscales of Neuro-QoL. Internal consistency reliability was evaluated by examining the item-total correlations and Cronbach’s alpha coefficients. The item-total correlations were calculated by removing each of the 24 items. We calculated and reported the alphas when any one of the items was removed from the instrument. Cronbach’s alpha was also reported for the whole instrument. For Cronbach’s alpha, we considered the following cut-off values: >0.7 (acceptable), >0.8 (good), and >0.9 (excellent). For item-total correlation, we considered a value greater than 0.3 to be an indicator that an item was related to the overall scale. Concurrent validity was tested by comparing other related scales. Stigma scores were calculated as a t-scores (50 is the mean and 10 is the standard deviation) (16).Patients with neurological diseases were consecutively recruited from the outpatient clinic of our neurology department. Six major neurological diseases were identified: relapsing-remitting multiple sclerosis (MS), Parkinson disease (PD), ischemic stroke, tension-type headache, generalized epilepsy, and chronic polyneuropathy. Literate patients were preidentified in terms of compliance to the test and those who scored 27 or higher on the Turkish version of the Mini Mental State Examination (MMSE-T) (17) were enrolled in the study. Sociodemographics and clinical characteristics including age, sex, education, employment status, marital status, and hospitalization were assessed.The Neuro-QoL stigma subscale consists of 24 items. Item scores range from 1 (never) to 5 . A summary index is calculated by adding all scores, ranging from 24 to 120, with higher scores reflecting the worst stigmatization (13).The World Health Organization’s Quality of Life (WHOQOL)-BREF with 26 items is a generic measure of health status comprising four major health dimensions , all of them on a scale of 26–135, with higher scores indicating full health status . In this study, the Turkish version of the WHOQOL-BREF was used (20).The Beck Depression Inventory (BDI) with 21 items is one of the popular depression measures. Scores are in the range of 0–63, with higher scores indicating severe depression. In this study, the Turkish version of the BDI was used (21–23).The Beck Anxiety Inventory (BAI) with 21 items is a severity indicator for anxiety. Scores are in the range of 0–63, with higher scores indicating severe anxiety (24). In this study, the Turkish version of the BAI was used (25).The General Self-Efficacy (GSE) scale measures the belief in one’s ability to complete activities related to one’s own competency. The GSE contains 17 items, 6 of which assess the level of positive self-esteem and 11 of which evaluate the level of negative self-esteem. Each item is scored from 1 (completely disagree) to 5 (completely agree) (26). In this study, the Turkish version of the GSE was used (27).The Multidimensional Scale of Perceived Social Support (MSPSS) is a research tool that assesses social support. It consists of three subscales, each of them representing a different source of support: family, friends, and significant other. It contains 12 items rated on a seven-point Likert-type scale with scores ranging from ‘very strongly disagree’ (= 1) to ‘very strongly agree’ (= 7) (28). In this study, the Turkish version of the MSPSS was used (29).Descriptive statistics were applied to demographic and questionnaire data. Internal consistency reliability was tested by Cronbach’s alpha indicator. Scores between 0.70 and 0.95 are considered as acceptable reliability indicators. Concurrent validity was tested by comparing the measured scores (one-way ANOVA with Tukey post hoc test for pairwise comparisons) among known group variables as well as by Pearson’s correlation coefficient. Pearson’s correlation coefficients were computed following the same criteria as above. P < 0.05 indicated statistical significance.Information about the research was given to all participants. The study protocol and ethics procedures were approved by the ethics board of our institution. Patients or their legal guardians provided informed signed consent.From a total of 152 patients, 65% were female and 35% were male. The mean age was 47.9 ± 17.6 years. The study group consisted of 29 patients with polyneuropathy, 25 patients with epilepsy, 23 patients with stroke, 24 patients with tension-type headache, 28 patients with MS, and 27 patients with PD.α results for each domain of the Neuro-QoL-Stigma scale. Table 2 shows the correlation matrix obtained and the significance values. Comparing Neuro-QoL-Stigma and other general measures, we found strong negative correlations with the GSE, MSPSS, and WHOQOL-BREF-T. We also found strong positive correlations with the BDI and BAI. We found no significant correlation with the family subdimension of the MSPSS. Table 1 shows the mean scores, the standard deviations, internal consistency correlations (ICCs), and Cronbach’s T-scores of the Neuro-QoL-Stigma scale for each group were 48.7 ± 11 for polyneuropathy, 49.1 ± 4.9 for epilepsy, 50.4 ± 11 for stroke, 45.2 ± 2.8 for headache, 51 ± 10.3 for MS, and 55.2 ± 12.5 for PD. When comparing our patients’ Neuro-QoL stigma T-scores with mean clinical T-scores of the Neuro-QoL study reference group , score difference of less than 0.5 SD units were determined within normal limits. When the groups were compared according to T-score, there was a significant difference between the headache and the PD group (P = 0.03).Stigma can be defined as a negative perception of chronically ill patients by their relatives or by society, or a similar self-perception by the patients themselves. Patients try to hide their illness from other people because of their feelings of embarrassment (30).Stigma is usually associated with neurological diseases as well as psychiatric illnesses so it is important to use a scale that can evaluate stigma for neurologists. With neurological diseases, stigma can lead to anxiety, depression, and decreased self-esteem and diminished life satisfaction. With the addition of long-term health problems, quality of life can be negatively affected (31). Our results showed a strong correlation with the scales  in support of the above information. Rao et al. developed a stigma scale for chronic disease. They described three types of stigmatization as follows: perceived stigma (discriminatory attitude by society), enacted stigma , and self-stigmatization  (7). Neuro-QoL Stigma also contains such areas.Various factors have been indicated in stigma studies in neurological diseases. In a stigma study performed among epilepsy patients, educational status, level of income, age at onset of the disease, and frequency of seizures were shown as influencing factors. Having adequate social support and increased self-efficacy has been found to have a positive effect on stigma scores. Increased knowledge of patients about epilepsy and the presence of positive attitudes towards epilepsy were associated with decreased stigma scores in patients with epilepsy . Victorson et al. reported that the T-score was 49.7 ± 9.1 in adult epilepsy patients by using the Neuro-QoL stigma scale (34). In the Neuro-QoL user manual, the T-score was defined as 50.6 ± 6.7 for epilepsy (16). We found the T-score for epilepsy to be 49.1 ± 4.9. This result is consistent with previous studies.PD patients have feelings of shame related to their movement and communication difficulties. Patients who cannot cope with social life due to their symptoms and who withdraw from society will have to live alone in their private world. Studies showed that higher stigma scores were related to more severe PD symptoms (35). Nowinski et al. showed that the stigma T-score was 48.39 ± 6.62 in PD cases (36). The Neuro-QoL user manual described the T-score as 49.29 ± 4.65 in PD (16). We found that the T-score for PD was 55.2 ± 12.5. Our results are higher than those of previous studies. This may be due to the possibly high level of disability in our PD cases. Further studies are required.MS is one of the most common causes of severe disability in young people. Apart from the neurological findings of MS, stigmatization, which affects the quality of life, must be considered by physicians. In stigma studies of MS, it was found that higher Expanded Disability Status Scale (EDSS) scores with higher age, longer disease duration, and progressive forms are found to be responsible for stigma . Miller et al. found average T-scores for MS cases as 49.3 ± 7.23 by using the Neuro-QoL short form (39). The Neuro-QoL user manual indicated the T-score as 50.13 ± 5.2 for MS (16). We found this score as 51 ± 10.3 for MS. Stroke-related stigma studies show that stroke survivors who experience mild-to-moderate levels of stigma are more likely to be depressed or have lower quality of life (40). The mean stigma T-score was 51.94 ± 6.33 for stroke patients in the Neuro-QoL user manual (16). We found the T-score as 50.4 ± 11 for stroke patients. It was found that tension-type headache is considerably more stigmatized than migraine in headache studies; patients may hide their symptoms and will not seek help or treatment (41). Young et al. investigated stigma in patients with episodic migraine, chronic migraine, and epilepsy. They observed that patients with chronic migraine and epilepsy had similar stigmatization. In their study, stigma correlated with inability to work (42). In the current study, we found the lowest T-score as 45.2 ± 2.8 in the headache group. Further studies are needed in different types of headache.In some polyneuropathy studies, it is found that some new developments have decreased the burden of stigmatism on patients and families over the past few decades. This may be due to recent medical treatments, primary care health professionals, and ongoing clinical trials (43). We found that the T-score was 48.7 ± 11 for the polyneuropathy group. Stigma may vary according to cultural differences, education level, and many other social and clinical factors. If patients cannot accept or understand their diagnosis, they cannot develop insight regarding their illness. This is the most important cause of self-stigmatization of patients (12). Stigma should be studied since it often accompanies neurological diseases. We aimed to evaluate the reliability and validity of the stigma scale of the Neuro-QoL tool. This scale can be used in clinical practice among different neurological diseases to understand stigmatization and it can be effective in treatment planning and prognosis of neurological diseases. All parameters of the adult long form of the Neuro-QoL-Stigma scale demonstrated high internal consistency and it is suitable for the Turkish population.


Risk estimation models integrated into Electronic Health Records (EHRs) can deliver innovative approaches in psychiatry, but clinicians' endorsement and their real-world usability are unknown. This study aimed to investigate the real-world feasibility of implementing an individualised, transdiagnostic risk calculator to automatically screen EHRs and detect individuals at-risk for psychosis.in-vitro phase (March 2018 to May 2018) and in-vivo phase (May 2018 to April 2019). The in-vitro phase addressed implementation barriers and embedded the risk calculator  into the local EHR. The in-vivo phase investigated the real-world feasibility of screening individuals accessing secondary mental healthcare at the South London and Maudsley NHS Trust. The primary outcome was adherence of clinicians to automatic EHR screening, defined by the proportion of clinicians who responded to alerts from the risk calculator, over those contacted.Feasibility implementation study encompassing an In-vitro phase: implementation barriers were identified/overcome with clinician and service user engagement, and the calculator was successfully integrated into the local EHR through the CogStack platform. In-vivo phase: 3722 individuals were automatically screened and 115 were detected. Clinician adherence was 74% without outreach and 85% with outreach. One-third of clinicians responded to the first email (37.1%) or phone calls (33.7%). Among those detected, cumulative risk of developing psychosis was 12% at six-month follow-up.This is the first implementation study suggesting that combining precision psychiatry and EHR methods to improve detection of individuals with emerging psychosis is feasible. Future psychiatric implementation research is urgently needed. This calculator has already demonstrated adequate external prognostic accuracy in two different independent EHRs   . Preventat scale . This caC = 0.79 ; n = 13, = 0.73) . Given t = 0.73) , these f = 0.73)  of this 2This study was approved by the East of England - Cambridgeshire and Hertfordshire Research Ethics Committee (Reference number: 18/EE/0066) and by the SLaM Caldicott Guardian. The protocol for this study was published  and is r2.1in-vitro”. This phase had two manifold aims: (i) to address implementation barriers according to the Consolidated Framework for Implementation Research (CFIR)   Fig. 2)in-vitro”i.e. lifespan-inclusive, individualised, clinically-based, transdiagnostic, automatic data acquisition and risk estimation, cheap, use of predictors widely available in clinical routine, usability in a sequential assessment framework) (a priori to match the CFIR “inner” and “outer” settings (vs retrospective EHR datasets). In the retrospective version of the risk calculator, individuals who developed psychosis within three months following their index diagnosis were excluded to mitigate for ICD-10 diagnostic instability. However, prospective implementation of this diagnostic lag was inefficient. Subsequent analyses confirmed that a refined version of the risk calculator without this lag period had similar external prognostic accuracy (Harrell's C = 0.79). We therefore used the refined model, optimised for prospective usability   were selsettings . The “adsettings , requirehttps://crisnetwork.co) (The CFIR “inner setting”  was charwork.co) . The CFIwork.co) .The CFIR “process” domain  was addrin-vitro phase focused on fine-tuning the use of the transdiagnostic risk calculator to the CFIR “individuals”/users' domain (https://ypmhag.org) to collect patients' feedback on practical and ethical issues relating to the real-world use of this risk calculator. We also conducted two group meetings with SLaM clinicians, to appraise barriers to clinicians' adherence and optimise sharing of recommendations made by the risk calculator.Most of the ' domain . We cons' domain  as well 2.22.2.1via CRIS), we started the in-vivo phase. During the study period (May 14th 2018 to April 29th 2019), all individuals (i) older than 14 years (ii) who were accessing any SLaM service (iii) receiving a first ICD-10 index primary diagnosis of any non-organic, non-psychotic mental disorder (eMethods 1), or a CHR-P designation and (iv) with existing contact details were deemed eligible. Clinicians were not required to enter any data; all predictors were recorded as part of clinical routine. During the study period, outreach was conducted with clinicians in Lambeth only, informing them about the study and the risk calculator. Clinicians in other boroughs only became aware of the study when they received the alerts to enable us to test the impact of outreach.Once the transdiagnostic risk calculator was embedded in the EHR  or at King's College London. If contact details of the responsible clinicians were incorrect, a third email was sent to the last clinician registered on EHR (e.g. their care coordinator) or to their GP. In a second step, if there was still no response following a further week, phone calls were initiated to the SLaM clinician or GP practice. Following discussion with the research team, the clinician then decided whether to formally initiate the referral—asking the patient if they consented to contact details being shared with the research team—or not. If the patient consented, they were contacted by the research team, and informed consent for face-to-face research was formally sought. Additional inclusion criteria were applied at this stage (eMethods 2). If an individual did not reach the threshold, the research team recommended no further assessment.Every week, all new individuals accessing SLaM who met eligibility criteria were automatically screened. If predictor data was missing, the calculator rechecked their availability each subsequent week, until the end of the study period. Although the original transdiagnostic risk prediction model can provide individualised estimates of psychosis risk up to a period of six years—with no predetermined thresholds and associated sensitivity or specificity—the primary aim of the 2.3The primary outcome was the adherence of clinicians to the use of the automatic EHR screening by the transdiagnostic risk calculator. This was operationalised as the proportion of clinicians who responded to recommendations of the calculator over those who were contacted by the research team. Secondary outcomes included: impact of different alerts on clinicians' adherence, the raw number of referrals initiated from secondary mental healthcare clinicians for an assessment of psychosis risk, and proportion of new ICD-10 diagnoses of psychotic disorders (eMethods 3) by six-month follow-up detected before their onset by the calculator across those screened.2.4t-tests; differences between categorical variables were assessed using two-tailed Fisher's exact test. For categorical variables with only two categories, an Odd's Ratio (OR) was calculated using Fisher's exact test. The cumulative incidence of psychosis was measured with Kaplan-Meier curves and 95% Greenwood confidence intervals  were assessed using independent sample two-tailed ntervals , and logntervals , and the33.1Implementation barriers were identified and overcome with clinician and service user engagement, and the calculator was successfully integrated into the local EHR through CogStack, an information retrieval and extraction platform for EHRs. The integration of the transdiagnostic risk calculator into the CogStack platform has been fully detailed in an associated publication . Clinici3.23.2.13722 patients presenting to SLaM clinical services during the study period and with data available in the EHR were eligible and automatically screened . 117 pat3.2.2For two patients, no clinician contact details were available on the EHR; 115 prompts were therefore sent to clinicians. Of these, 89 clinicians (77.4%) responded to prompts sent on the recommendation of the transdiagnostic risk calculator.3.2.3p = .03). Clinicians' response to the prompts increased from 74.1%  to 85.3% (29/34 in Lambeth) when outreach was deployed, but this difference was non-significant . Among the 89 patients for whom clinicians responded, 18 (20.2%) patients were excluded (eResults 3). Of the remaining 71 patients, 39 (54.9%) were referred by their responsible clinician to OASIS for a face-to-face assessment. The predominant reason for non-referral was patients experiencing acute phases of psychiatric symptom severity which required intensive care either in inpatient units or the community, and therefore were unable to undergo a research assessment. Other reasons for non-referral are presented in eResults 4. Among those screened (n = 3722), 3640 (97.79%) were followed-up through the EHR, and 38 (1.04%) developed a psychotic disorder by six-month follow-up. The cumulative incidence of psychosis in those screened was 0.016  at six-months .33 clinicians (37.1%) responded to the first email, 20 (22.5%) to the second, six (6.7%) to the third and 30 (33.7%) responded to phone calls. Including patient names in SLaM emails instead of citing Trust IDs  raised the response rate from 37.5% (15/40) to 58.7% (44/75) , 101 (87.82%) were followed-up through the EHR and nine (8.9%) developed a psychotic disorder by six-months. The cumulative incidence of psychosis in those detected was 0.12  at six-months , which was significantly higher than in those screened (log-rank test: p < 0.001).Among those detected .Among the 49 patients detected but not referred  and with a six-month follow-up in the EHR, three (6.1%) developed a psychotic disorder. The cumulative incidence of psychosis in those detected but not referred was 0.147  at six-months  and comparable to that observed in those detected and referred  prognostic models in this field. While other individualised risk calculators are available (e.g. NAPLS)  , these a. NAPLS) .in-vitro and in-vivo), may represent a viable method to overcome the implementation gap, which has led some to question the utility of precision psychiatry  or labour (e.g. cognitive tasks) to produce their predictions. Because the current risk calculator is simple, it can be used to screen large populations, with more complex    or costlre, PPS) .e.g. Natural Language Processing, NLP)   or addinng, NLP) . Secondlng, NLP) . Interesng, NLP) . It is tng, NLP) .https://www.pronia.eu/) and PSYSCAN  (MC_PC_16048) to PF-P. DO is supported by the UK Medical Research Council (MR/N013700/1) and King's College London member of the MRC Doctoral Training Partnership in Biomedical Sciences. MB, HB, RS and RD are part-funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at the South London and Maudsley NHS Foundation Trust and King's College London. RP has received support from an MRC Health Data Research UK Fellowship (MR/S003118/1) and a Starter Grant for Clinical Lecturers (SGL015/1020) supported by the Academy of Medical Sciences, The Wellcome Trust, MRC, British Heart Foundation, Arthritis Research UK, the Royal College of Physicians and Diabetes UK.This study was supported by the King's College London Confidence in Concept award from the PF-P has received advisory consultancy fees from Lundbeck outside of this work. RS has received research support from Roche, Janssen, GSK and Takeda outside of this work. The authors have declared that there are no conflicts of interest in relation to the subject of this study.


Lycopene is the most abundant carotenoid in tomatoes, which has been identified to have the properties of anti-inflammation in addition to the capability to inhibit the expression of adhesion molecules. Intercellular adhesion molecules play a critical role in the pathogenesis of psoriasis. Here, we report that the topical use of a lycopene decreased imiquimod (IMQ)-induced psoriasis-like inflammatory responses, the progress of which was based on adhesion molecules. In vitro analysis showed that lycopene decreased keratinocyte and monocyte adhesion. Evidence suggests that intercellular adhesion molecule-1 (ICAM-1) is a main mediator of psoriasis pathogenesis. Therefore, it will be interesting to investigate the factors that contribute to the lycopene-mediated inhibition of ICAM-1 expression in psoriasis. We expect that lycopene will with potential value in the treatment of psoriasis. Psoriasis is a chronic inflammatory skin disease that is characterized by the formation of scaly, erythematous plaques. Psoriasis affects approximately 2% of the population . The patSK-Hep-1 cells [Lycopene is a hydrocarbon phytochemical that belongs to the tetraterpene carotenoid family and is found in red vegetables and fruits, such as watermelon, pink guava, and tomato. Lycopene has been found to be active in many cell lines, including prostate cancer and oral cancer cell lines . Lycopen-1 cells . Lycopen-1 cells . Another-1 cells . In addi-1 cells . In stom-1 cells . Therefo-1 cells . Althoug-1 cells . AccordiDuring the experimental period, there were not significant differences in the final weight and weight gain of the animals (data not shown) among the groups. As shown in The representative photos A show psTo further study the effects of lycopene on epidermal proliferation, hematoxylin and eosin (HE)-staining were used to analyze the structural features characteristic of animals′ IMQ-induced psoriasis-like skin. In TNF-α simultaneously increased the adhesion of keratinocytes and monocytes, which plays a key role in psoriasis. Therefore, we performed an adhesion assay to study the effect of lycopene on TNF-α-stimulated keratinocyte-monocyte adhesion. Nutraceuticals are natural, nutritional compounds that can be efficacious in preventative medicine or in the treatment of disease. Several foods, such as fruits, vegetables, cereals, and legumes  supplemeAtopic dermatitis is a long-term type of inflammation of the skin. It results in itchy, red, swollen, and cracked skin. Oral administration of lycopene prevents atopic dermatitis in hairless mice . AnotherAdhesion molecules include intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecule-1 (VCAM-1) and have a key role in the inflammatory response . The forw/w) was purchased from INOVA Pharmaceutical Co. . Lycopene was purchased from Rexall Sundown, Inc.  for animal studies and purchased from Sigma-Aldrich Co.  for in vitro studies. The molecular structure of lycopene is shown in IMQ cream . The study procedures conformed to the “Guide for the Care and Use of Laboratory Animals” . 2 on the shaved back for 7 consecutive days. All the animals were randomly divided into six groups  and housed in microisolator cages on a 12-h day/night cycle with water ad libitum. Thirty 6- to 8-week-old male C57BL/6 mice  were used. The psoriasis-like skin inflammation model was established according to a previous reference with modification . Mice wes n = 5; B. Group Blood were collected from animals for biochemical assays before entering the experiment, at the end of 6th week, and at sacrifice. The blood were extracted from the mandibular artery into sodium citrate-containing tubes. Plasma AST, ALT, BUN, TG, and total cholesterol were analyzed using the automatic chemistry system .The animals were sacrificed at the end of 5th week. The back skin was collected and rinsed with ice-cold phosphate buffered saline; fixed by 4% paraformaldehyde, and embedded in paraffin. Then, 5-µm cross-sections were stained with H&E staining. Slides were observed via the TissueGnostics TissueFAXS and HistoFAXS System . 2 contained incubator. When performing the experiments, the HaCaT cells were grown to 90% confluence.The HaCaT cell was cultured in Dulbecco′s modified Eagle′s medium supplemented with antibiotics (100 U/m penicillin A and 100 U/mL streptomycin), fetal bovine serum (10%), and glutamine (2 mM). Cells were kept in a 37 °C humidified and 5% CO5) were seeded into 24-well plates before the assay. Then, the growth medium was supplemented with TNF-α at 5 ng/mL for 24 h. THP1 cells were labeled with 10 μM of 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester  for 1 h at 37 °C in serum-free RPMI 1640 medium; the cells were then washed with PBS to remove the free dye and resuspended in RPMI 1640 containing 2% FBS. One million labeled THP-1 cells were added to each HCAEC-containing well, and the incubation continued for 1 h. The non-adherent cells were removed by three gentle washes with HBSS. The number of THP-1 cells adhered to the HaCaT cells was observed using inverted fluorescence microscopy and counted using a Multilabel Counter Victor2  at an emission wavelength of 530 nm and an absorption wavelength of 435 nm after the cells were lysed with DMSO.The HaCaT cells  and anti-ICAM-1  antibodies. The loading control was used as an anti-β-actin  antibody. The proteins were visualized using an enhanced chemiluminescence detection kit .p < 0.05 was considered as statistically significant. The results are expressed as the mean ± SEM. ANOVA followed by Dunnett′s test was used to analyze the data.


Legionella dumoffii can be inhibited by Galleria mellonella apolipophorin III (apoLp-III) which is an insect homologue of human apolipoprotein E., and choline-cultured L. dumoffii cells are considerably more susceptible to apoLp-III than bacteria grown without choline supplementation. In the present study, the interactions of apoLp-III with intact L. dumoffii cells cultured without and with exogenous choline were analyzed to explain the basis of this difference. Fluorescently labeled apoLp-III (FITC-apoLp-III) bound more efficiently to choline-grown L. dumoffii, as revealed by laser scanning confocal microscopy. The cell envelope of these bacteria was penetrated more deeply by FITC-apoLp-III, as demonstrated by fluorescence lifetime imaging microscopy analyses. The increased susceptibility of the choline-cultured L. dumoffii to apoLp-III was also accompanied by alterations in the cell surface topography and nanomechanical properties. A detailed analysis of the interaction of apoLp-III with components of the L. dumoffii cells was carried out using both purified lipopolysaccharide (LPS) and liposomes composed of L. dumoffii phospholipids and LPS. A single micelle of L. dumoffii LPS was formed from 12 to 29 monomeric LPS molecules and one L. dumoffii LPS micelle bound two molecules of apoLp-III. ApoLp-III exhibited the strongest interactions with liposomes with incorporated LPS formed of phospholipids isolated from bacteria cultured on exogenous choline. These results indicated that the differences in the phospholipid content in the cell membrane, especially PC, and LPS affected the interactions of apoLp-III with bacterial cells and suggested that these differences contributed to the increased susceptibility of the choline-cultured L. dumoffii to G. mellonella apoLp-III.The growth of  Legionellaceae family, which are ubiquitous in water and soil environments. Legionella species can survive in all elements of systems supplying water from the natural sources to the tap. Legionella spp. contaminates water sources such as air conditioning, cooling towers, whirlpool spas, as well as medical and industrial equipment function simultaneously as amplifiers or disseminators of these bacteria. The bacteria can be inhaled into the lungs, where they invade alveolar macrophages. The capability of Legionella spp. to intracellularly proliferate in immune cells (designed to kill bacteria), and therefore use them as host cells, is a prerequisite for development of the disease. In Europe, 5500 to 6500 cases of Legionnaires’ disease are reported annually [Legionellosis is an important but uncommon bacterial respiratory infection with varying severity, i.e., from the flu-like infection called Pontiac fever which does not require specialized treatment, to acute multilobar pneumonia called Legionnaires’ disease which can result in death. Humans can be infected in both the living environment  and during a hospital stay . The infection may have a sporadic or outbreak form ,2. The dannually . In the annually . The higLegionellaceae family have been isolated from both natural and clinical sources (http://www.bacterio.cict.fr/l/legionella.html). In the USA and Europe, the most common cause of legionellosis was L. pneumophila, which has been responsible for approximately 90% of culture-confirmed cases. Serogroup 1 was predominant (84.2%), and serogroups 2–3 (7.4%) accounted for the remaining cases. However, based on isolation from patients, L. dumoffii has been regarded as one of 28 non-L. pneumophila species that have been reported to be pathogenic in humans [L. pneumophila species, L. dumoffii causes about 10% of Legionnaires’ disease cases, which are often fatal, especially in immunocompromised patients [Legionella spp. [L. dumoffii is responsible for pericarditis, prosthetic valve endocarditis, and septic arthritis [To date, 65 species of bacteria from the oups 2–3 .4% accoudominant 4.2%, andrthritis ,12.Legionella cell and its phagotrophic host. L. dumoffii does not produce a capsule or an exopolysaccharide. Therefore, the predominant molecule presented on the cell surface of these bacteria is the lipopolysaccharide (LPS). On the one hand, LPS functions as a non-fimbrial adhesin, which determines colonization of host cells, and thus becomes an important factor of Legionella virulence involved in the complex mechanisms of the disease development. On the other hand, LPS is an efficient barrier that protects bacteria from environmental factors, drugs, and hydrophobic substances [An efficiently functioning bacterial envelope is essential for communication between the bstances . LPS locbstances .Legionella spp. cell membranes [L. pneumophila with the host cell [Legionella spp. can synthesize PC from exogenous choline [L. dumoffii, structurally different PC species are localized in both the inner and outer membranes. Interestingly, L. dumoffii cultured in the presence of exogenous choline exhibits a reduced capability of TNF-α induction, which enables the bacteria to evade the host immune system [Phosphatidylcholine (PC), i.e., a phospholipid that is a common membrane component of eukaryotic cells, also constitutes an important part of embranes . The preost cell . It has  choline ,18,19,20Galleria mellonella bound to the cell surface of different bacteria and fungi, which was accompanied by antimicrobial action of the protein [L. pneumophila and L. dumoffii to antibacterial action of G. mellonella apoLp-III [L. pneumophila cell envelope taking place during binding to the bacterial cell surface [L. pneumophila LPS and caused alterations in the L. pneumophila cell surface [The lipid-binding protein, apolipophorin III (apoLp-III) is an insect homologue of human apolipoprotein E (apoE) engaged in lipid transport, as well as immune response in insects. The protein is an exchangeable component of lipophorin particles. Similarly to the apoE molecule, whose N-terminal domain forms a bundle of four α-helices in an up-and-down spatial arrangement, the apoLp-III molecule is a bundle of five antiparallel α-helices, in which hydrophobic chains are directed to the interior and hydrophilic chains are exposed to the environment ,22. In a protein ,30,31,32poLp-III ,34,35. O surface .L. pneumophila, L. dumoffii cells cultured in the presence of exogenous choline were considerably more susceptible, that is, they showed three times lower survival rate, after treatment with G. mellonella apoLp-III at a concentration of 0.4 mg/mL, than the bacteria grown without supplementation of this compound [Legionella [L. dumoffii grown on choline-supplemented medium exhibited a ca. 12% increase in the content of PC in favor of phosphatidylethanolamine (PE) as compared with bacteria cultured without exogenous choline [L. dumoffii cells grown with and without choline supplementation. In addition, fluorescence lifetime imaging microscopy (FLIM) measurements revealed that apoLp-III interacted with considerably higher affinity and penetrated more deeply the liposomes formed of lipids extracted from choline-cultured L. dumoffii, i.e., containing a higher PC level [Unlike compound . These rgionella ,19 suggegionella . Indeed, choline . FurtherPC level .L. dumoffii grown with and without choline supplementation or whether these differences were mainly due to changes in the composition of the outer membrane phospholipids caused by choline supplementation. To elucidate the molecular basis of the higher susceptibility of choline-grown L. dumoffii to G. mellonella apoLp-III, apoLp-III interactions with intact L. dumoffii cells cultured without and with exogenous choline were assessed in the present study. The effects of apoLp-III binding to the bacterial cells on their surface topography and nanomechanical properties were analyzed by atomic force microscopy (AFM). The binding ability of apoLp-III to the bacterial cells was compared by using fluorescently labeled apoLp-III and fluorescence lifetime imaging microscopy (FLIM). The characterization of the L. dumoffii LPS was performed for the first time. Formation of L. dumoffii LPS and apoLp-III complexes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. Furthermore, a detailed analysis of apoLp-III interactions with the phospholipids and LPS of the L. dumoffii cell envelope were performed on a liposomal model using FLIM. The use of PLs and LPS isolated from non- and choline-supplemented L. dumoffii allowed analysis of apoLp-III interactions with systems that reflected the natural layout containing various PLs species, in contrast to models based on defined components.The aim of this work was to analyze whether the presence of LPS had an impact on the documented differences in the interaction of apoLp-III with L. dumoffii cell surface was decorated with very small, mostly regular granules, which were well visible on the bacterial height maps  in the cell surface of the bacteria grown without the choline addition. No such recesses were detected in the cell surface of the bacteria cultured on the choline-supplemented medium .L. dumoffii  were incubated without or in the presence of apoLp-III  at the concentration 0.1 mg/mL, and then analyzed by AFM. The results are presented as ±SD. The differences between two mean values were established using the U Mann–Whitney’s test.L. dumoffii cells. The cell envelope elasticity was affected most severely by apoLp-III. ApoLp-III caused a ca. six-fold increase in the cell surface elasticity in the bacteria grown without choline in contrast to the choline-cultured L. dumoffii whose elasticity did not change after the incubation with apoLp-III. Although the cell surface roughness and adhesion forces seemed not to be affected by the culture conditions, they both were influenced by the apoLp-III treatment (Growth in the presence of exogenous choline resulted in a ca. two-fold increase in the Derjaguin–Muller–Toporov (DMT) modulus, reflecting elasticity, which indicated a change in the nanomechanical properties of the cell envelope. Furthermore, considerable alterations in the cell surface properties resulted from the apoLp-III treatment of reatment .L. dumoffii cells cultured on the medium with and without choline showed that the bacteria produced vesicles, which were present on the cell surfaces .The SEM images and X-ray analysis showed that L. dumoffii cells, as demonstrated by fluorescence microscopy imaging (The fluorescently labeled apolipophorin III (FITC-labeled apoLp-III) bound to the  imaging . ImportaL. dumoffii cells, incorporation of FITC-apoLp-III to the lipid membrane environment increased considerably the fluorescence lifetime, i.e., from 3.2 ns in the water phase to 31.1 ns [L. dumoffii cells. 1 = 0.53 ns, τ2 = 1.6 ns, τ3 = 4 ns, and τ4 = 11 ns. Legionella cells cultured with choline supplementation, there is a long-lifetime component (τ4 = 11 ns) that has not been detected in the control  acids was dominated by amide-linked 3-hydroxy acids (L. dumoffii contained 18 3-hydroxy fatty acids in the range of C12–C21 and 8 non-hydroxylated acids (C14–C17); 3-hydroxy fatty acids were dominated by n3-OH 14:0 (10%), a3-OH 18:0 (9%), and n3-OH 16:0 (8%). Long chain (ω-1)-oxo fatty acid (27-oxo-octacosanoic) and -dioic acids  were also found in the L. dumoffii LPS; 27-oxo-octacosanoic acid was the major fatty acid in the LPS.Fatty acids in the LPS preparations were analyzed with GLC-MS of methyl esters. The fatty acid profile including 26 both linear and methyl branched-chain . During the SEC analyses in physiological saline (PBS), the L. dumoffii LPS obtained from both bacteria supplemented with choline and those grown without supplementation gave single peaks eluting at 10.70 and 10.57 min, respectively . In turn, the percentage of the free form of protein for LPS from bacteria grown without choline supplementation is 60.6%. At the 1:50 mass ratio of protein and LPS from choline-supplemented bacteria, 18.2% of apoLp-III occurs in the free form, while the percentage of the free form of protein for LPS isolated from L. dumoffii grown without choline supplementation accounts for 11.8%. To confirm the formation of a complex between apoLp-III and LPS micelles at the 1:50 mass ratio, the fraction containing the LPS micelle peak eluting at 10.87 min .Additionally, the incubation of apoLp-III with higher amounts of LPS causes a gradual decrease in the apoLp-III peak, suggesting that the protein forms complexes with 0.87 min C,D was c0.87 min D. The bag et al. . Taking L. dumoffii cells cultured in the presence (+Chol) and absence (−Chol) of exogenous choline and giant unilamellar vesicles (GUV) formed with DPPC. Lipids isolated from bacteria cultured on the medium with choline contained more PC (12%) and less phosphatidylethanolamine (approximately the same range) as compared with lipids from bacteria cultured on the medium without choline supplementation [L. dumoffii cell envelope, purified L. dumoffii LPS was used for formation of SUVs, in addition to L. dumoffii PLs. ApoLp-III was labeled with FITC which enabled examination of binding of the protein to lipid membranes by means of fluorescence lifetime imaging microscopy (FLIM). L. dumoffii LPS, recorded after exposure to apoLp-III. As can be seen, the interaction of the protein with membranes formed of lipids extracted from cells cultured in the presence of exogenous choline and containing LPS as an additional component of liposomes, resulted in apparent degradation of lipid membranes, manifested by the liposome fragmentation. A liposome model was applied to examine the interaction of apoLp-III with lipid membranes revealed the following: Small unilamellar vesicles (SUV) formed with the lipid components of entation . To resoL. dumoffii LPS  and, as a consequence, affected the interactions with apoLp-III. Interestingly, Verdon et al. [L. pneumophila tolerance to the antimicrobial peptide warnericin RK, resulting from alterations in the composition and length of fatty acids in phospholipid molecules. Previous studies have demonstrated the susceptibility of entation ,35. We amembrane . A detaibacteria . These rg et al.  on apoLpg et al. . Probabln et al.  reportedL. dumoffii, LPS purified from these bacteria was used in the present study. We demonstrated that apoLp-III can form stable complexes with L. dumoffii LPS. Furthermore, the presence of L. dumoffii LPS in GUVs formed from DPPC considerably increased the interaction of apoLp-III with these liposomes. Interestingly, the incorporation of the LPS into SUVs composed of PLs from the choline-supplemented L. dumoffii resulted in fragmentation of the liposomes upon the interaction with apoLp-III. This indicates that LPS is an important component of the L. dumoffii cell envelope which facilitates apoLp-III binding; however, it is the differences in the PC content that are mainly responsible for stronger interaction of apoLp-III with the membrane, and consequently, for the higher susceptibility of choline-cultured bacteria to G. mellonella apoLp-III.In order to resolve the role of LPS in apoLp-III interactions with L. pneumophila and its eukaryotic host. L. pneumophila mutants deficient in PC formation are less virulent than the respective wild type in their interaction with the host [G. mellonella apoLp-III with L. dumoffii cells. The increased content of PC in the cell membrane promoted apoLp-III binding to bacterial cells which, in consequence, increased the susceptibility of L. dumoffii to apoLp-III. It can be hypothesized that PC, which is important for interactions with the host and for establishing a replicative niche in its cell, also plays a role of “a receptor” for potential antibacterial molecules, for example, apoLp-III. However, binding of such molecules to PC could mask the presence of this phospholipid on the bacterial surface and impede the interaction of the bacteria with the host cell. Consequentially, this would prevent the establishment of a replicative niche and intracellular development of the bacteria. Moreover, since the choline-cultured L. dumoffii with increased PC content in the cell membrane exhibited reduced capability of TNF-α induction [It is known that PC plays an important role in the interaction between the host . Our resnduction , such PCL. pneumophila produces and releases OMVs from the cell envelope. Extramembrane vesicles, containing virulence-associated proteins, consist of a double lipid membrane, which, similar to the outer membrane of bacteria, is composed of LPS localized in the outer leaflet and PLs in the inner leaflet [L. dumoffii cultured on the medium with and without exogenous choline can produce OMVs. Our study of the apoLp-III interaction with the components of the L. dumoffii cell envelope was performed on a model of liposomes (SUVs) formed of PLs with incorporated LPS isolated from the bacteria. Such liposomes mimic L. dumoffii OMVs. Therefore, our results suggest that this type of vesicles can be destroyed upon apoLp-III treatment. This could be considered to be a possible way to fight against Legionella and provides a new interesting idea for the development of alternative anti-Legionella therapies.Similar to other Gram-negative bacteria,  leaflet ,44. The  leaflet . In addi leaflet . In the Legionella dumoffii ATCC 33279  was cultured for 3 days at 37 °C on buffered charcoal yeast agar (BCYE) medium  or on a medium enriched with 100 µg/mL choline chloride . Bacteria cultured on the BCYE medium with and without exogenous choline were harvested with 0.5 M NaCl and centrifuged at 8000× g for 20 min. The cell pellets were washed once with 0.5 M NaCl and once with distilled water and lyophilized. Approximately 2 g of bacterial mass were obtained from L. dumoffii cultured with and without choline and subjected to isolation of phospholipids (PLs) and lipopolysaccharide (LPS). L. dumoffii responsible for the blue and white autofluorescence under long-wavelength UV light [PLs were isolated from the bacterial mass according to the Bligh and Dyer method, as previously described . In thisUV light . Phosphog, 4 °C, 4 h), and freeze-dried. The LPS species distributed between the phenol and water phases as hydrophobic and hydrophilic fractions accounted for, respectively, approximately 2% and 1% of the dry mass of bacteria cultured on both the choline-supplemented and non-supplemented medium. The LPSs were further analyzed to determine their fatty acid and sugar composition. LPS from the phenol phase, due to its higher yield, was used for formation of liposomes and investigation of interactions with apoLp-III.Delipidated and dried cell pellets were suspended in 50 mM phosphate buffer (pH 7.0) supplemented with 5 mM EDTA and digested with lysozyme . Nucleic acids were digested by RNAse and DNAse , and then polypeptides were hydrolyzed with proteinase K . LPS was extracted from digested cells three times with aq 45% phenol at 68 °C . The LPSSalmonella enterica serovar Typhimurium  was used as a standard.The LPSs isolated from bacterial masses harvested from the medium with or without choline were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, 4.8% and 12% polyacrylamide-bisacrylamide gels were used as the stacking and separating gels, respectively. LPS (2 µg) was mixed with a sample buffer  in a 1:1 ratio (v/v). LPS bands were visualized using the silver staining method . LPS froFor fatty acid analysis, an LPS sample (1 mg) was subjected to hydrolysis in 4 M HCl . Free fatty acids were extracted with chloroform, and then converted into methyl esters by methanolysis . Then, the solution was cooled to room temperature and the solvent was evaporated. Fatty acid methyl esters recovered by extraction with chloroform/water  were trimethylsilylated with HMDS/TMCS/pyridine   and analyzed using gas liquid chromatography and mass spectrometry (GLC-MS).For neutral and amino sugar analysis, the LPS samples were hydrolyzed with 2 M trifluoroacetic acid (TFA) , N-acetylated, reduced with NaBD4, and acetylated with a 1:1 (v/v) pyridine-acetic anhydride mixture . The products were identified as alditol acetates by GLC-MS.The methyl esters of fatty acids and the alditol acetates of monosaccharides were analyzed by GLC-MS using a gas chromatograph  connected to an MSD 5975C (inert XL EI/Cl) detector. The chromatograph was equipped with an HP-5ms (SLB-5ms) capillary column . A temperature gradient of 150 °C (5 min) to 310 °C, at 5 °C/min, was applied, and the final temperature was kept for 10 min.G. mellonella , as described in detail in our previous study [G. mellonella hemolymph and methanolic extracts was determined according to Bradford [Apolipophorin III was purified from hemolymph of us study . In brieBradford .The procedure of apoLp-III labeling with fluorescein isothiocyanate  was described in detail in our previous paper . BrieflyL. dumoffii LPS and apoLp-III was verified by size exclusion chromatography (SEC) using a Superdex 200 10/300 GL column  and an UltiMate 3000 HPLC system . The separations were carried out at room temperature (RT) in 50 mM sodium phosphate pH 7.4 containing 150 mM NaCl (PBS buffer), in PBS buffer supplemented with 1% (w/v) (SDS) or in PBS buffer containing 6 M guanidinum hydrochloride (GuaHCl). The flow rate was 0.7 mL/min, the spectrophotometric detection was carried out at 215 and 280 nm, and the column was calibrated using the following molecular mass standards : Blue Dextran 2000 (2 MDa), thyroglobulin (669 kDa), ferritin (443 kDa), catalase (232 kDa), aldolase (157 kDa), conalbumin (75 kDa), ovoalbumin (43 kDa), RNAse (13.7 kDa), and tryptophan (204 Da). Estimated total and void volumes of the column and retention volumes of standard proteins were used for calculation of distribution coefficients (KAV) and plotted versus the logarithm from the molecular masses of standard proteins. Linear regression of the obtained curve was used for calculation of molecular masses of compounds resolved during chromatography. All apoLp-III and LPS mixtures, as well as their individual solutions, were pre-incubated in PBS buffer, for 1 h, at 37 °C, immediately before injection on the column in order to facilitate formation of the protein-LPS complex. Additionally, before separations, the samples analyzed in denaturing conditions (PBS buffer containing 1% SDS) were mixed with the SDS stock solution to 3% (w/v) SDS final concentration and boiled for 5 min. In turn, the samples separated in the chaotropic buffer (PBS buffer with 6 M GuaHCl) were mixed with solid GuaHCl to a final concentration of 6 M and preincubated for 15 min at RT. Fractions eluting from the column and containing the LPS-apoLp-III complex were manually collected, concentrated on a 3 kDa cut-off centrifugal ultrafilter , resolved in reducing conditions using SDS-PAGE [The formation of the complex between SDS-PAGE , and eleL-α-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamineB sulfonyl)  were purchased from Sigma-Aldrich  and Avanti Polar Lipids, Inc. , respectively.L. dumoffii LPS.Giant Unilamellar Vesicles (GUVs) were formed from DPPC with 1 mol% of rhodamine B-labeled lipid. Constituents of the lipid phase of liposomes were deposited onto two platinum electrodes (35 × 4 × 0.5 mm) by evaporation from the solution, under a stream of gaseous nitrogen. Residuals of organic solvents were removed during incubation for 2–3 h under vacuum. Platinum electrodes with deposited lipid films were fixed in a Teflon holder at a distance of 3 mm. The electrodes were placed in a cuvette containing a buffer solution . Electric connections were attached to an AC field supply (DF 1641A). GUV electroformation was carried out over 2 h with an applied AC sinusoidal field with 10 Hz frequency and voltage of 9 V (peak-to-peak). The temperature during the electroformation was stabilized at 45 °C . GUVs containing LPS were formed as described above with addition of 2 mol% of purified L. dumoffii cells cultured without and with choline supplementation were used for formation of small unilamellar vesicles (SUV). Briefly, chloroform and methanol solutions of lipids , were mixed to attain a homogeneous solution. The solvents were evaporated under a stream of nitrogen and the lipids with LPS or lipids without LPS film of the test tube were dried under vacuum for 2–3 h. A MQ water solution  was added to the dried film at about 35 °C and vortexed vigorously for 5 min. Next, the lipids were sonicated in ice for 3 sec with 60% amplitude and titanium probe using a VCX-130 sonicator , and then heated to 35 °C. The sonication-heating cycles were repeated 3 times. Liposomes were prepared twice and FLIM imaging of at least 5 selected liposomes was performed from each series.Lipids (2 mg/mL) extracted from The liposome suspensions (20 µL) were deposited on a microscope slide. Then, 2 µL of FITC-labeled apoLp-III (0.0074 µg of protein) were added and the liposomes were immediately imaged and analyzed with FLIM spectroscopy.The experiments were performed using a two-channel MicroTime 200  confocal system coupled to an Olympus IX71 inverted microscope. The instrument was equipped with a piezo-scanner objective working in an 80 × 80 μm maximum imaging range at a nominal positioning accuracy of 1 nm. The sample was illuminated with a 470 nm pulsed laser focused on the objects of interest by a water immersion objective . A 75 μm diameter confocal pinhole was used. In the case of the GUV studies, the fluorescence beam was split by 50% reflecting mirror and observed simultaneously by two analyzers  in separate spectral windows. One of them detected the fluorescence of FITC (by a 520/35 dichroic band-pass filter) and the other analyzer detected rhodamine (by a 580/70 band-pass filter). Both filters were provided by Chroma-AHF AnalysenTechnik. The SUV samples were studied using a single detector in a 520/35 spectral window. The microscopy system recorded fluorescence intensity and lifetime simultaneously. The intensity decays were analyzed by fitting an exponential model using SymPhoTime 64 v. 2.4 software .600 = 0.2. The suspension was mounted on an aluminum plate and left to dry at room temperature. The samples were imaged by scanning electron microscopy (SEM).Bacteria cultured on the BCYE medium with and without choline supplementation were suspended in water to achieve optical density ODThe elemental analysis of the surface of bacterial cells and outer membrane vesicles (OMV) was performed using a FEI Quanta 3D FEG scanning electron microscope . Energy dispersive X-ray (EDX) microanalysis was performed using a silicon drift detector (SDD) operating at 30 kV acceleration voltage. The microanalysis was carried out without any shadowing and the concentrations of the elements were determined using the standardless method.L. dumoffii cells  grown without and with exogenous choline supplementation were incubated with FITC-apoLp-III  at 37 °C, for 5 or 15 min. Then, the bacterial suspensions were centrifuged  and washed three times with 20 mM phosphate buffer, pH 7.4, containing 0.9% NaCl. Finally, the bacteria were suspended in 20 mM phosphate buffer pH 7.4 (10 µL) and bound to polylysine coated microscopic slides for imaging [L. dumoffii grown on the choline-supplemented and non-supplemented medium, integrated density was calculated from 40 images obtained from two independent experiments using ImageJ software ver. 1.440 (rsb.info.nih.gov/ij/). Integrated density is defined as the sum of the light of pixels in the image and reflects the light intensity of the bacterial cells.For fluorescence imaging,  imaging . The bacL. dumoffii cells cultured without or with exogenous choline (OD600 = 0.01) were incubated with FITC-apoLp-III , for 1.5 h, at 37 °C. After incubation, the bacterial suspensions were spun down , the supernatant was removed, and the cells were gently washed once with 200 µL of sterile 10 mM phosphate buffer pH 7.4 and twice with 200 µL of non-pyrogenic water, and finally centrifuged as described above. Afterwards, the bacteria were suspended in 80 µL of non-pyrogenic water. For FLIM measurements, 10 µL of the resulting suspension was applied on the surface of a cover slip coated with polylysine and carefully rinsed three times with water after 30 sec. The FLIM measurements were carried out using a confocal MicroTime 200 system  coupled to an OLYMPUS IX71 microscope. The samples were excited with a pulsed laser at 470 nm (repetition rate 5 MHz and time resolution 16 ps) and the photons were collected with a 60× water immersed objective . The restricted effective confocal volume was 0.26 fL. Scattered light was removed by a ZT 473RDC XT band pass interference filter  and the observation was carried out with the use of 495 and 488 nm long-wavelength-pass filters. A single photon sensitive avalanche photodiode (τ-SPAD) was used for collection of fluorescence photons and processing was accomplished by the HydraHarp400 time-correlated single photon counting (TCSPC) module. SymPhoTime 64 software package (v. 1.6) was used for analysis of the data.For fluorescence lifetime imaging microscopy (FLIM), 100 µL of a water suspension containing L. dumoffii cells (OD600 = 0.2) cultured on the choline-supplemented or non-supplemented BCYE medium were incubated, for 1 h, at 37 °C, without (control) and with apoLp-III . Then, the bacterial suspensions were centrifuged at 8000× g for 10 min, at 4 °C, and prepared on mica discs, as described in our previous paper [L. dumoffii prepared on the mica discs was imaged using a NanoScope V AFM . The measurements were carried out in the “Peak Force QNM” operation mode using a TAP150A Antimony doped Si tip with a spring constant of 1.646 N·m−1 . During each of the two independent experiments, three randomly chosen fields were imaged on each mica disc. The data obtained were analyzed with Nanoscope Analysis ver. 1.40 software . The values that define nanomechanical properties were calculated from 40 fields (340 × 340 nm) measured over the entire bacterial cell surface in 3 × 3 µm areas. The differences between two mean values were established using the U Mann–Whitney’s test. The section profiles and three-dimensional (3D) images of the cells were generated using WSxM 5.0 software  [For atomic force microscopy (AFM) imaging, 4 × 40 μL of water suspension containing us paper . The cel, Spain) .